Bottinelli R, Coviello D A, Redwood C S, Pellegrino M A, Maron B J, Spirito P, Watkins H, Reggiani C
Institute of Human Physiology, University of Pavia, Italy.
Circ Res. 1998;82(1):106-15. doi: 10.1161/01.res.82.1.106.
Mutant contractile protein genes that cause familial hypertrophic cardiomyopathy (FHC) are presumed to encode mutant proteins that interfere with contractile function. However, it has generally not been possible to show mutant protein expression and incorporation into the sarcomere in vivo. This study aimed to assess whether a mutant alpha-fast tropomyosin (TM) responsible for FHC is actually expressed and determines abnormal contractile function. Since alpha-fast TM is expressed in heart and skeletal muscle, samples from vastus lateralis muscles were studied from two FHC patients carrying an Asp175Asn alpha-fast TM mutation and two healthy control subjects. TM isoforms from whole biopsy samples and single fibers were identified by gel electrophoresis and Western blot analysis. An additional faster-migrating TM band was observed in both FHC patients. The aberrant TM was identified as the Asp175Asn alpha-fast TM by comigration with purified recombinant human Asp175Asn alpha-fast TM. Densitometric quantification of mutant and wild-type alpha-fast TMs suggested equal expression of the two proteins. Contractile parameters of single skinned muscle fibers from FHC patients and healthy control subjects were compared. Calcium sensitivity was significantly increased in muscle fibers containing Asp175Asn alpha-fast Tm compared with fibers lacking the mutant TM. No discernible difference was found regarding cooperativity, maximum force, and maximum shortening velocity. This is the first demonstration that the mutant TM that causes FHC is indeed expressed and almost certainly incorporated into muscle in vivo and does result in altered contractile function; this confirms a dominant-negative, rather than null allele, action. Since the mutant TM was associated with increased calcium sensitivity, this mutation might be associated with an enhancement and not a depression of cardiac contractile performance. If so, this contrasts with the hypothesis that FHC mutations induce contractile impairment followed by compensatory hypertrophy.
导致家族性肥厚型心肌病（FHC）的突变收缩蛋白基因被认为编码的突变蛋白会干扰收缩功能。然而，一般来说，在体内无法显示突变蛋白的表达以及其整合到肌节中。本研究旨在评估导致FHC的突变α-快肌钙蛋白（TM）是否实际表达，并确定其是否导致异常的收缩功能。由于α-快TM在心脏和骨骼肌中表达，因此对两名携带Asp175Asnα-快TM突变的FHC患者和两名健康对照者的股外侧肌样本进行了研究。通过凝胶电泳和蛋白质印迹分析鉴定了全活检样本和单根肌纤维中的TM同工型。在两名FHC患者中均观察到一条额外的迁移速度更快的TM条带。通过与纯化的重组人Asp175Asnα-快TM共迁移，将异常TM鉴定为Asp175Asnα-快TM。对突变型和野生型α-快TM的光密度定量分析表明这两种蛋白表达量相等。比较了FHC患者和健康对照者单根去表皮肌纤维的收缩参数。与缺乏突变TM的肌纤维相比，含有Asp175Asnα-快Tm的肌纤维的钙敏感性显著增加。在协同性、最大力量和最大缩短速度方面未发现明显差异。这是首次证明导致FHC的突变TM确实表达，几乎可以肯定其在体内整合到肌肉中，并确实导致收缩功能改变；这证实了其显性负性作用，而非无效等位基因作用。由于突变TM与钙敏感性增加有关，这种突变可能与心脏收缩性能的增强而非减弱有关。如果是这样，这与FHC突变诱导收缩功能受损继而发生代偿性肥大的假说形成对比。
